ProMET

Workpackages

WP1: Characterisation of heterogeneous protein material at the primary structure level

WP leader: NML at LGC

Other participants: BAM, CNR, CNRS, GUF, INRIM, LNE, LUMC, MHRA, NPL, PTB, TUBITAK, UCL

Objective: The aim of WP1 is to investigate the influence of primary sequence variants and PTMs of proteins, such as glycosylation or phosphorylation, on the assessment of the chemical purity. Protein materials of increasing complexity will be produced and characterised by expanding conventional metrological approaches based on MS and NMR.

Structure – the WP is divided into 6 Tasks:

  • Task 1.1: Sourcing and production of protein models
  • Task 1.2: Structurally-related impurity identification in recombinant protein materials
  • Task 1.3: Structurally-related impurity quantification
  • Task 1.4: SI-traceable quantification of PTMs site occupancy
  • Task 1.5: Quantification of glycosylated protein
  • Task 1.6: Purity assessment of antibody

WP2: HOS of proteins, characterisation and quantification

WP leader: CNRS

Other participants: BAM, GUF, INRIM, NML at LGC, LNE, MHRA, NPL, PTB, UCL

Objective: The aim of WP2 is to investigate the influence of the primary sequence and PTMs variants considered in Objective 1 on the secondary and tertiary structure of the protein and the influence of the presence of different structure variants (from primary to tertiary) on protein-protein interactions. This will be achieved by developing fit-for-purpose strategies using native and structural MS (e.g. ion mobility spectrometry-MS (IM‑MS), hydrogen deuterium exchange MS (HDX‑MS), chemical cross-linking MS (XL‑MS)) combined with other biophysical approaches (e.g. dynamic light scattering (DLS), cryoEM, and NMR) or existing computational techniques to characterise the higher-order structure (HOS) of the proteins to distinguish and quantify different structures and to explore the protein-protein interactions observed in biological systems (e.g. antibody-antigen interactions). Protocols to ensure traceability and estimation of the measurement uncertainties of the results will be developed.

Structure – the WP is divided into 4 Tasks:

  • Task 2.1: HDX-MS metrological assessment
  • Task 2.2: IM-MS analytical performances and assessment of new generation instrumentation for tandem ion mobility
  • Task 2.3: Complementary biophysical methods for protein conformational dynamics assessment
  • Task 2.4: Structural characterisation of protein-protein Interactions

WP3: Assessing the impact of protein primary and HOS on current quantification strategies

WP leader: PTB

Other participants: BAM, CNR, CNRS, INRIM, NML at LGC, LNE, MHRA, TUBITAK, UCL

Objective: The aim of WP3 is to study the impact of structure heterogeneities from objective 1 and 2 on the measurement procedures using either purified peptides, recombinant proteins, extracted proteins or endogenous proteins in simplified buffer or matrix-matched solutions as calibrators. The influence of structure and interactions on isotope dilution-based reference measurement procedures (RMPs) and routine methods using proteomics approaches and immunoassays, including sample preparation, calibration and incorporation of isotopically labelled analogues, will be investigated.

Structure – the WP is divided into 4 Tasks:

  • Task 3.1: Assessing the impact of different primary protein structural forms on the calibration of immunoassay and MS based methods
  • Task 3.2: Assessing the impact of secondary and higher order protein structural differences
  • Task 3.3: Investigating the behaviour of protein variants on sample preparation methods when using isotope dilution approaches
  • Task 3.4: Observation and assessment of differences in quantitative measurement results provided by antibody and mass spectrometry based methods

WP4: Metrology framework for the assessment of the influence of protein variations on their function

WP leader: BAM

Other participants: CNR, CNRS, GUF, INRIM, NML at LGC, LNE, LUMC, MHRA, NPL, PTB, TUBITAK, UCL

Objective: WP4 aims to determine mathematical models based on the results of Objectives 1 to 3 to understand the influence of interferences, protein structure and protein-protein interactions on the different measurement procedures and evaluate their influence on protein function. Approaches to estimate the appropriate measurement uncertainty associated with procedures targeting entities that are not the intended measurand will be developed. Based on the measurement results and the model outcomes, guidelines will be developed for the definition of protein measurands (including appropriate units), of the analytical targets and the estimation of measurement uncertainty for the routine methods and the method developed within the project.

Structure – the WP is divided into 4 Tasks:

  • Task 4.1: Development of approaches for the determination of protein function
  • Task 4.2: Data compilation and correlation
  • Task 4.3: Evaluation of measurement uncertainty
  • Task 4.4: Guidelines for the definition of protein measurands, analytical targets, and the estimation of measurement uncertainty

WP5: Creating Impact

WP Leader: LUMC

Other participants: BAM, CNR, CNRS, GUF, INRIM, NML at LGC, LNE, MHRA, NPL, PTB, TUBITAK, UCL

Objective: The aim is to maximise the impact of this project within the European community of stakeholders and industrial end-users, including those within other EURAMET countries outside EMPIR. This will ensure that this project achieves the highest possible impact on the European region.

Structure – the WP is divided into 2 Tasks:

  • Task 5.1: Dissemination and communication
  • Task 5.2: Exploitation and uptake